Calcium/calmodulin (Ca2+/CaM) dependent protein kinases (CaM-kinases) are important signal transduction enzymes which are particularly abundant in brain. In brain and other tissues they are crucial in regulating numerous physiological functions including neurotransmitter synthesis and release, several ion channels and gene expression. This proposal will examine important structure/function and regulatory features of two CaM-dependent kinases, CaM-kinase II and CaM-kinase IV. These studies will utilize purified enzymes expressed in the baculovirus/Sf9 cell system. Techniques of protein chemistry, site-specific mutagenesis and enzymology will be employed. Major components of these studies will include: 1. Detailed analysis of the functionality of the autoinhibitory domain of CaM-kinase II, its regulation by autophosphorylation, and its interaction with the catalytic domain. These results will be used as a biochemical basis for molecular modeling of CaM-kinase II. These studies will clarify the regulatory model for this enzyme which has important consequences for its involvement as a long-term sensor of synaptic activity. 2. The structure and regulation of CaM-kinase IV and its substrate specificity will be determined. Particular emphasis will be placed on its enzymatic properties which may be important for Ca2+ -dependent transcriptional regulation of immediate early genes. 3. Attempts will be made to obtain crystals of CaM-kinase II which would be suitable for subsequent X-ray determination of higher ordered structure. These studies will further our understanding of the structural, regulatory and enzymatic properties of these signal transduction enzymes, thereby furthering our understanding of their probable physiological roles in learning and memory, epilepsy and stroke.